The present invention relates to a method for high spatial resolution stochastic examination of a sample structure labeled with a substance, whereby a biological structure is used as the structure labeled with the substance, molecules of the substance may be present in a first state and in a second state, and the first and second states differ from one another in at least one photophysical property.
Methods for high spatial resolution stochastic examination of a sample structure labeled with a substance are known in the art. In this regard, reference is made to WO 2006/127692 A2, US 2008/0032414 A1 and WO 2007/128434 A1, which describe stochastic high-resolution and localization microscopy methods, which are known as PALM, STORM, PALMIRA and GSDIM, respectively. Moreover, it is known from the journal “Nature Methods”, Vol. 5, No. 2, February 2008, pp. 155-157, that photoactivated localization microscopy (PALM) can be performed on living cells, making it possible to track movements of individual particles or molecules. This method is also referred to as sptPALM (single particle tracking PALM).
In all of the known methods for high spatial resolution stochastic examination of a sample structure labeled with a substance, where a biological structure is used as the structure labeled with the substance, molecules of the substance may be present in a first state and in a second state. These first and second states differ from one another in at least one photophysical property. This photophysical property often consists in the ability to fluoresce. In other words, there may be a first fluorescent state and a second non-fluorescent state.
In the known methods, a substance with which a structure of interest is labeled is actively switched between two states. In PALM, PALMIRA or STORM, for example, the substance is switched from a non-fluorescent state to a fluorescent state. This is usually accomplished using light. In this process, care is taken to ensure that only so many molecules of the substance are in the fluorescent state, so that most of the signals that are detected by a microscope having a CCD camera, for example, can be uniquely associated with individual molecules. This is the fundamental idea underlying these stochastic methods, in which a plurality of images are recorded. After determining the centroid of the recorded signals, a high-resolution image is constructed from the centroids by superimposing the plurality of recorded images.
In the GSDIM method, for example, a fluorescent substance is “pumped” into a dark state A, in which it does not fluoresce and from which it may spontaneously return to the initial fluorescent state B. Using a defined illumination, it can be achieved that only a certain amount of molecules fluoresce simultaneously, and can thus be localized. Analogously to the other high-resolution stochastic methods, a high-resolution image can be generated by recording a series of images of signals from single molecules and determining and summing the individual centroids.
All of the previously known methods for high spatial resolution stochastic examination of a sample structure labeled with a substance require active switching between two states. This switching is typically accomplished by light, for example by illumination with a laser. The need for this active switching operation makes the known methods complex.